The detailed analysis of the microbiome and resistome of artisanal blue-veined cheeses provides evidence on sources and patterns of succession linked with quality and safety traits.

BACKGROUND: Artisanal cheeses usually contain a highly diverse microbial community which can significantly impact their quality and safety. Here, we describe a detailed longitudinal study assessing the impact of ripening in three natural caves on the microbiome and resistome succession across three different producers of Cabrales blue-veined cheese. RESULTS: Both the producer and cave in which cheeses were ripened significantly influenced the cheese microbiome. Lactococcus and the former Lactobacillus genus, among other taxa, showed high abundance in cheeses at initial stages of ripening, either coming from the raw material, starter culture used, and/or the environment of processing plants. Along cheese ripening in caves, these taxa were displaced by other bacteria, such as Tetragenococcus, Corynebacterium, Brevibacterium, Yaniella, and Staphylococcus, predominantly originating from cave environments (mainly food contact surfaces), as demonstrated by source-tracking analysis, strain analysis at read level, and the characterization of 613 metagenome-assembled genomes. The high abundance of Tetragenococcus koreensis and Tetragenococcus halophilus detected in cheese has not been found previously in cheese metagenomes. Furthermore, Tetragenococcus showed a high level of horizontal gene transfer with other members of the cheese microbiome, mainly with Lactococcus and Staphylococcus, involving genes related to carbohydrate metabolism functions. The resistome analysis revealed that raw milk and the associated processing environments are a rich reservoir of antimicrobial resistance determinants, mainly associated with resistance to aminoglycosides, tetracyclines, and ß-lactam antibiotics and harbored by aerobic gram-negative bacteria of high relevance from a safety point of view, such as Escherichia coli, Salmonella enterica, Acinetobacter, and Klebsiella pneumoniae, and that the displacement of most raw milk-associated taxa by cave-associated taxa during ripening gave rise to a significant decrease in the load of ARGs and, therefore, to a safer end product. CONCLUSION: Overall, the cave environments represented an important source of non-starter microorganisms which may play a relevant role in the quality and safety of the end products. Among them, we have identified novel taxa and taxa not previously regarded as being dominant components of the cheese microbiome (Tetragenococcus spp.), providing very valuable information for the authentication of this protected designation of origin artisanal cheese. Video Abstract.

Designing multitarget ligands for neurodegenerative diseases with improved permeability trough PLGA nanoencapsulation.

Multitarget ligands (MTLs) have emerged as an interesting alternative for addressing complex multifactorial pathologies such as neurodegenerative diseases. However, a common challenge associated with these compounds is often their high molecular weight and low solubility, which becomes a hurdle when trying to permeate over the blood-brain barrier (BBB). In this study, we have designed two new MTLs that modulate three pharmacological targets simultaneously (tau, beta-amyloid and TAR DNA-binding protein 43). To enhance their brain penetration, we have formulated organic polymeric nanoparticles using poly(lactic-co-glycolic acid). The characterization of the formulations, evaluation of their permeability through an in vitro BBB model, and assessment of their activity on disease-representative cellular models, such as Alzheimer's disease and amyotrophic lateral sclerosis, have been conducted. The results demonstrate the potential of the new MTLs and their nanoparticle encapsulation for the treatment of neurodegenerative diseases.

Nanomedical research and development in Spain: improving the treatment of diseases from the nanoscale.

The new and unique possibilities that nanomaterials offer have greatly impacted biomedicine, from the treatment and diagnosis of diseases, to the specific and optimized delivery of therapeutic agents. Technological advances in the synthesis, characterization, standardization, and therapeutic performance of nanoparticles have enabled the approval of several nanomedicines and novel applications. Discoveries continue to rise exponentially in all disease areas, from cancer to neurodegenerative diseases. In Spain, there is a substantial net of researchers involved in the development of nanodiagnostics and nanomedicines. In this review, we summarize the state of the art of nanotechnology, focusing on nanoparticles, for the treatment of diseases in Spain (2017-2022), and give a perspective on the future trends and direction that nanomedicine research is taking.

PROTAC-Induced Glycogen Synthase Kinase 3ß Degradation as a Potential Therapeutic Strategy for Alzheimer's Disease.

Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine kinase and an attractive therapeutic target for Alzheimer's disease. Based on proteolysis-targeting chimera (PROTAC) technology, a small set of novel GSK-3ß degraders was designed and synthesized by linking two different GSK-3ß inhibitors, SB-216763 and tideglusib, to pomalidomide, as E3 recruiting element, through linkers of different lengths. Compound 1 emerged as the most effective PROTAC being nontoxic up to 20 µM to neuronal cells and already able to degrade GSK-3ß starting from 0.5 µM in a dose-dependent manner. PROTAC 1 significantly reduced the neurotoxicity induced by Aß25-35 peptide and CuSO4 in SH-SY5Y cells in a dose-dependent manner. Based on its encouraging features, PROTAC 1 may serve as a starting point to develop new GSK-3ß degraders as potential therapeutic agents.

Susceptibility and transcriptomic response to plasma-activated water of Listeria monocytogenes planktonic and sessile cells.

Plasma-Activated Water (PAW) was generated from tap water using a surface dielectric barrier discharge at different discharge power (26 and 36 W) and activation time (5 and 30 min). The inactivation of a three-strain Listeria monocytogenes cocktail in planktonic and biofilm state was evaluated. PAW generated at 36 W-30 min showed the lowest pH and the highest hydrogen peroxide, nitrates, nitrites contents and effectiveness against cells on planktonic state, resulting in 4.6 log reductions after a 15-min treatment. Although the antimicrobial activity in biofilms formed on stainless steel and on polystyrene was lower, increasing the exposure time to 30 min allowed an inactivation >4.5 log cycles. The mechanisms of action of PAW were investigated using chemical solutions that mimic its physico-chemical characteristics and also RNA-seq analysis. The main transcriptomic changes affected carbon metabolism, virulence and general stress response genes, with several overexpressed genes belonging to the cobalamin-dependent gene cluster.

The background microbiota and sanitization agent drive the fate of Listeria monocytogenes in multispecies biofilms formed on a plasma-polymerized coating applied on stainless steel.

The present study evaluates the anti-biofilm activity of a coating applied with an atmospheric-pressure plasma jet system on AISI 316 stainless steel (SS) against multispecies biofilms containing Listeria monocytogenes (using background microbiota from three different meat industries) using culture-dependent and culture-independent approaches. Also, the disinfection effectiveness and biofilm evolution after sanitization with two food industry biocides were assessed. The anti-biofilm activity of the coating against L. monocytogenes, observed on mono-species biofilms (p < 0.05), was lost on the multispecies biofilms developed for 7 days at 12 °C (p > 0.05), with L. monocytogenes counts ranging from 5.5 ± 0.7 to 6.1 ± 0.5 CFU/cm2 on the uncoated SS and from 4.4 ± 0.2 to 6.4 ± 0.5 CFU/cm2 on the coated SS. The taxonomic composition of the formed biofilms was highly dependent on the industry but not affected by the artificial inoculation with L. monocytogenes and the nature of the surface (coated vs uncoated SS). When L. monocytogenes was artificially inoculated, its growth was partially controlled in the biofilms developed, with the magnitude of this effect being lower (p < 0.05 on coated SS) for the industry with the lowest taxonomy richness and diversity (3.8 ± 0.2 CFU/cm2), as compared the other two sampled industries (2.4 ± 0.4 and 1.6 ± 0.2 CFU/cm2). The 15-min disinfection treatments with either sodium hypochlorite or peracetic acid at 0.5 % resulted in total viable and L. monocytogenes counts below the limit of detection in most cases, immediately after treatment. The subsequent incubation of the sanitized plates for another 7 days at 12 °C in fresh BHI media led to the development of biofilms with lower bacterial richness and alpha diversity, and higher beta diversity. Even though sodium hypochlorite was in general slightly less effective than peracetic acid immediately after application, it caused a stronger growth control (p < 0.05) of the naturally present L. monocytogenes on the multispecies biofilms developed. This finding highlights the importance of understanding the interspecific competitive relationships between the members of the background microbiota and L. monocytogenes for the long-term control of this pathogen in food processing facilities.

Plasma-activated water: A cutting-edge technology driving innovation in the food industry.

Innovation regarding food production and processing is required to meet the emerging challenges of ensuring worldwide food security and meeting consumer demands for high-quality, safe and nutritious food products. This review provides insights into the current state-of-the-art of the emerging applications of Plasma Activated Water (PAW) in the food industry. PAW antimicrobial properties, inactivation mechanisms and the critical factors determining the lethal effect, as well as the bases for other technological applications are discussed. Overall, this review article describes the degree of success achieved by PAW technology in different applications and illustrates its feasibility and applicability in the food-processing industry.

Development and characterization of anti-biofilm coatings applied by Non-Equilibrium Atmospheric Plasma on stainless steel.

Biofilm-mediated microbial persistence of pathogenic and spoilage bacteria is a serious problem in food industries. Due to the difficulty of removing mature biofilms, great efforts are being made to find new strategies to prevent bacterial adherence to surfaces, the first step for biofilm development. In this study, coatings of (3-aminopropyl)triethoxysilane (APTES), tetraethyl orthosilicate (TEOS) and acrylic acid (AA) were applied by Non-Equilibrium Atmospheric Plasma on stainless steel (SS) AISI 316, the SS most commonly used in food industry equipment. Their anti-biofilm activity was assessed against Listeria monocytogenes CECT911 and Escherichia coli CECT515 after incubation at 37 °C. The best results were obtained for L. monocytogenes, with coatings consisting of a base coating of APTES and a functional coating of TEOS (AP10 + TE6) or AA (AP10 + AA6) that reduced biofilm production by 45% and 74%, respectively, when compared with the uncoated SS. These coatings were further characterized, together with a variation of the best one that replaced the acrylic acid with succinic acid (AP10 + SA6). Their anti-biofilm activity was assessed under different incubation conditions, including two strains of L. monocytogenes isolated from processing environments of a meat industry. The coating AP10 + AA6 reduced the biofilm formation by 90% after incubation at 12 °C, a temperature more representative of those commonly found in food processing environments. The morphological and physico-chemical characterization of the selected coatings showed that the coating with the highest anti-biofilm activity (i.e., AP10 + AA6) had lower surface roughness and higher hydrophilicity. This suggests that the formation of a hydration layer prevents the adherence of L. monocytogenes, an effect that seems to be enhanced by low temperature conditions, when the wettability of the strains is increased.

Durability Assessment of a Plasma-Polymerized Coating with Anti-Biofilm Activity against L. monocytogenes Subjected to Repeated Sanitization.

Biofilm formation on food-contact surfaces is a matter of major concern causing food safety and spoilage issues to this sector. The aim of this study was to assess the durability of the anti-biofilm capacity of a plasma-polymerized coating composed of a base coating of (3-aminopropyl)triethoxysilane (APTES) and a functional coating of acrylic acid (AcAc). Coated and uncoated AISI 316 stainless steel (SS) plates were subjected to five sanitization cycles with sodium hypochlorite (0.05%) and peracetic acid (0.5%). The effectiveness of the coating for the inhibition of multi-strain Listeria monocytogenes biofilm formation was confirmed using a three-strain cocktail, which was grown on the SS plates at 12 °C for 6 days. Compared to the uncoated SS, relative biofilm productions of 14.6% on the non-sanitized coating, 27.9% on the coating after sanitization with sodium hypochlorite, and 82.3% on the coating after sanitization with peracetic acid were obtained. Morphological and physicochemical characterization of the coatings suggested that the greater anti-biofilm effectiveness after sanitization with sodium hypochlorite was due to the high pH of this solution, which caused a deprotonation of the carboxylic acid groups of the functional coating. This fact conferred it a strong hydrophilicity and negatively charged its surface, which was favorable for preventing bacterial attachment and biofilm formation.

Microbial colonization and resistome dynamics in food processing environments of a newly opened pork cutting industry during 1.5 years of activity.

BACKGROUND: The microorganisms that inhabit food processing environments (FPE) can strongly influence the associated food quality and safety. In particular, the possibility that FPE may act as a reservoir of antibiotic-resistant microorganisms, and a hotspot for the transmission of antibiotic resistance genes (ARGs) is a concern in meat processing plants. Here, we monitor microbial succession and resistome dynamics relating to FPE through a detailed analysis of a newly opened pork cutting plant over 1.5 years of activity. RESULTS: We identified a relatively restricted principal microbiota dominated by Pseudomonas during the first 2 months, while a higher taxonomic diversity, an increased representation of other taxa (e.g., Acinetobacter, Psychrobacter), and a certain degree of microbiome specialization on different surfaces was recorded later on. An increase in total abundance, alpha diversity, and ß-dispersion of ARGs, which were predominantly assigned to Acinetobacter and associated with resistance to certain antimicrobials frequently used on pig farms of the region, was detected over time. Moreover, a sharp increase in the occurrence of extended-spectrum ß-lactamase-producing Enterobacteriaceae and vancomycin-resistant Enterococcaceae was observed when cutting activities started. ARGs associated with resistance to ß-lactams, tetracyclines, aminoglycosides, and sulphonamides frequently co-occurred, and mobile genetic elements (i.e., plasmids, integrons) and lateral gene transfer events were mainly detected at the later sampling times in drains. CONCLUSIONS: The observations made suggest that pig carcasses were a source of resistant bacteria that then colonized FPE and that drains, together with some food-contact surfaces, such as equipment and table surfaces, represented a reservoir for the spread of ARGs in the meat processing facility. Video Abstract.

Heterogeneity in biofilm formation and identification of biomarkers of strong biofilm formation among field isolates of Pseudomonas spp.

The biofilm formation ability of a collection of thirty-three Pseudomonas spp. isolates from food processing facilities was investigated in order to find biomarkers of strong biofilm production, a characteristic that can determine persistence in food processing environments. The strains were classified according to the colony pigmentation on solid media as green, brown or not pigmented. The biofilm production on stainless steel and polystyrene was assessed by spectrometric determination of the fixed crystal violet, and the biofilm formed on glass, through confocal laser scanning microscopy. Besides, pyoverdine production, catalase activity, RpoS status and cellular hydrophobicity were also monitored. A significantly higher biofilm production level on stainless steel and polystyrene was observed for green-pigmented strains as compared to brown or not pigmented strains. The influence of iron availability on biofilm formation on stainless steel was studied through the addition of the iron scavenger 2,2-bipyridine resulting in a decrease of 40 % in biofilm formation for the not pigmented strains. For most of the potential biomarkers studied (i.e., pyoverdine production, catalase activity, cellular hydrophobicity), the phenotypic heterogeneity observed among strains was mainly dependent on the Pseudomonas species and no strong associations with the biofilm formation capacity were detected. However, the green colony pigmentation on solid media showed good potential as a biomarker of strong biofilm formation on stainless steel and polystyrene both in P. aeruginosa and Pseudomonas spp.

The role of the general stress response regulator RpoS in Cronobacter sakazakii biofilm formation.

The relationship between biofilm formation and RpoS status was assessed in nine field isolates of C. sakazakii. Their ability to form biofilms was studied in BHI and minimum media with different pH values and supplemented or not with the amino acids arginine, lysine and glutamic acid. Biofilm formation, both on polystyrene and stainless steel, was measured by spectrometric determination of the fixed crystal violet and the biofilms were visualized by confocal laser scanning microscopy and scanning electron microscopy. Despite the existing heterogeneity among the different strains, biofilm formation was generally higher in buffered minimum media (pH 7.0) supplemented with lysine than in other culture media and on stainless steel plates than on polystyrene. The results showed a lower ability to form biofilms for a strain with a loss-of-function mutation in the rpoS gene, the general stress response regulator of Gram-negative bacteria, when compared to the rest of the strains, which harboured a functional rpoS. The complementation of this strain with a functional rpoS gene resulted in an increase in its biofilm formation ability up to levels comparable to those observed for strains with a functional rpoS. However, the differences were markedly reduced when the incubation time was increased from 24 to 48 h, indicating that the loss of RpoS caused a delay in the development of mature biofilms, rather than a complete inhibition of biofilm production in C. sakazakii.